Masaki Arioka

Acceleration of bone regeneration by local application of lithium: Wnt signal-mediated osteoblastogenesis and Wnt signal-independent suppression of osteoclastogenesis.

Inhibition of glycogen synthase kinase (GSK)-3 and the consequent activation of the Wnt/β-catenin signaling pathway have been reported to increase bone volume. To develop a novel pharmacotherapy for injured bone, we investigated whether GSK-3 inhibitor was effective in promoting bone formation. In in vitro experiments, we examined the effects of GSK-3 inhibitors LiCl and SB216763 on osteoblastogenesis of mesenchymal progenitor C3H10T1/2 cells and osteoclastogenesis of osteoclast precursor RAW-D cells. Both inhibitors promoted osteoblast differentiation, assessed by alkaline phosphatase activity and calcium deposition, stimulating the Wnt/β-catenin signaling pathway and thereby inducing Runx2. On the other hand, the GSK-3 inhibitors suppressed osteoclast differentiation, assessed by tartrate-resistant acid phosphatase staining and number of nuclei in the cells, reducing NFATc1 expression independently of the Wnt/β-catenin signaling pathway. In subsequently performed in vivo studies, we examined the effect of locally administered Li2CO3 on the recovery from a partial defect made on the rat tibia. Computerized tomography and bone histomorphometry showed that Li2CO3 accelerated bone regeneration in defect lesion with increased lamellar bone ratio compared with the controls. These results suggested that local application of lithium (or other GSK-3 inhibitors) might effectively facilitate recovery from bone injury by promoting osteoblastogenesis and inhibiting osteoclastogenesis.

Acceleration of bone development and regeneration through the Wnt/β-catenin signaling pathway in mice heterozygously deficient for GSK-3β.

Glycogen synthase kinase (GSK)-3β plays an important role in osteoblastogenesis by regulating the Wnt/β-catenin signaling pathway. Therefore, we investigated whether GSK-3β deficiency affects bone development and regeneration using mice heterozygously deficient for GSK-3β (GSK-3β(+/-)). The amounts of β-catenin, c-Myc, cyclin D1, and runt-related transcription factor-2 (Runx2) in the bone marrow cells of GSK-3β(+/-) mice were significantly increased compared with those of wild-type mice, indicating that Wnt/β-catenin signals were enhanced in GSK-3β(+/-) mice. Microcomputed tomography of the distal femoral metaphyses demonstrated that the volumes of both the cortical and trabecular bones were increased in GSK-3β(+/-) mice compared with those in wild-type mice. Subsequently, to investigate the effect of GSK-3β deficiency on bone regeneration, we established a partial bone defect in the femur and observed new bone at 14 days after surgery. The volume and mineral density of the new bone were significantly higher in GSK-3β(+/-) mice than those in wild-type mice. These results suggest that bone formation and regeneration in vivo are accelerated by inhibition of GSK-3β, probably through activation of the Wnt/β-catenin signaling pathway.

Stage‐dependent benefits and risks of pimobendan in mice with genetic dilated cardiomyopathy and progressive heart failure

The Ca2+ sensitizer pimobendan is a unique inotropic agent that improves cardiac contractility with less of an increase in oxygen consumption and potentially fewer adverse effects on myocardial remodelling and arrhythmia, compared with traditional inotropes. However, clinical trials report contradictory effects of pimobendan in patients with heart failure (HF). We provide mechanistic experimental evidence of the efficacy of pimobendan using a novel mouse model of progressive HF.

Celecoxib inhibits osteoblast maturation by suppressing the expression of Wnt target genes

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to impair bone healing. We previously reported that in colon cancer cells, celecoxib, a COX-2-selective NSAID, inhibited the canonical Wnt/β-catenin signaling pathway. Since this pathway also plays an important role in osteoblast growth and differentiation, we examined the effect of celecoxib on maturation of osteoblast-like cell line MC3T3-E1. Celecoxib induced degradation of transcription factor 7-like 2, a key transcription factor of the canonical Wnt pathway. Subsequently, we analyzed the effect of celecoxib on two osteoblast differentiation markers; runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), both of which are the products of the canonical Wnt pathway target genes. Celecoxib inhibited the expression of both RUNX2 and ALP by suppressing their promoter activity. Consistent with these observations, celecoxib also strongly inhibited osteoblast-mediated mineralization. These results suggest that celecoxib inhibits osteoblast maturation by suppressing Wnt target genes, and this could be the mechanism that NSAIDs inhibit bone formation and fracture healing.

2,5-Dimethylcelecoxib prevents pressure-induced left ventricular remodeling through GSK-3 activation.

Glycogen synthase kinase-3 (GSK-3) is a crucial regulator of cardiac hypertrophy. We previously reported that 2,5-dimethylcelecoxib (DM-celecoxib), a celecoxib derivative unable to inhibit cyclooxygenase-2, prevented cardiac remodeling by activating GSK-3, resulting in lifespan prolongation in a mouse model of genetic dilated cardiomyopathy. In the present study, we investigated whether DM-celecoxib can also prevent pressure-induced cardiac remodeling and heart failure, elicited by transverse aortic constriction (TAC). Before testing the effects of DM-celecoxib, we compared the effects of TAC on the hearts of wild-type and GSK-3β hetero-deficient (GSK-3β+/−) mice to determine the role of GSK-3 in cardiac remodeling and heart failure. GSK-3β+/− mouse hearts exhibited more severe hypertrophy, which was characterized by accelerated interstitial fibrosis, than wild-type mouse hearts after TAC, suggesting that reduced GSK-3β activity aggravates pressure-induced left ventricular remodeling. We subsequently examined the effects of DM-celecoxib on TAC-induced cardiac remodeling. DM-celecoxib inhibited left ventricular systolic functional deterioration, and prevented left ventricular hypertrophy and fibrosis. It also activated GSK-3α and β by inhibiting Akt, suppressing the activity of β-catenin and nuclear factor of activated T-cells and thereby decreasing the expression of the Wnt/β-catenin target gene products fibronectin and matrix metalloproteinase-2. These results suggest that DM-celecoxib is clinically useful for treating pressure-induced heart diseases.

Celecoxib and 2,5-dimethylcelecoxib inhibit intestinal cancer growth by suppressing the Wnt/β-catenin signaling pathway

We previously reported that celecoxib, a selective COX‐2 inhibitor, strongly inhibited human colon cancer cell proliferation by suppressing the Wnt/β‐catenin signaling pathway. 2,5‐Dimethylcelecoxib (DM‐celecoxib), a celecoxib analog that does not inhibit COX‐2, has also been reported to have an antitumor effect. In the present study, we elucidated whether DM‐celecoxib inhibits intestinal cancer growth, and its underlying mechanism of action. First, we compared the effect of DM‐celecoxib with that of celecoxib on the human colon cancer cell lines HCT‐116 and DLD‐1. 2,5‐Dimethylcelecoxib suppressed cell proliferation and inhibited T‐cell factor 7‐like 2 expression with almost the same strength as celecoxib. 2,5‐Dimethylcelecoxib also inhibited the T‐cell factor‐dependent transcription activity and suppressed the expression of Wnt/β‐catenin target gene products cyclin D1 and survivin. Subsequently, we compared the in vivo effects of celecoxib and DM‐celecoxib using the Mutyh−/− mouse model, in which oxidative stress induces multiple intestinal carcinomas. Serum concentrations of orally administered celecoxib and DM‐celecoxib elevated to the levels enough to suppress cancer cell proliferation. Repeated treatment with celecoxib and DM‐celecoxib markedly reduced the number and size of the carcinomas without showing toxicity. These results suggest that the central mechanism for the anticancer effect of celecoxib derivatives is the suppression of the Wnt/β‐catenin signaling pathway but not the inhibition of COX‐2, and that DM‐celecoxib might be a better lead compound candidate than celecoxib for the development of novel anticancer drugs.

Anti-tumor effects of differentiation-inducing factor-1 in malignant melanoma: GSK-3-mediated inhibition of cell proliferation and GSK-3-independent suppression of cell migration and invasion

ABSTRACT Differentiation‐inducing factor‐1 (DIF‐1) isolated from Dictyostelium discoideum strongly inhibits the proliferation of various mammalian cells through the activation of glycogen synthase kinase‐3 (GSK‐3). To evaluate DIF‐1 as a novel anti‐cancer agent for malignant melanoma, we examined whether DIF‐1 has anti‐proliferative, anti‐migratory, and anti‐invasive effects on melanoma cells using in vitro and in vivo systems. DIF‐1 reduced the expression levels of cyclin D1 and c‐Myc by facilitating their degradation via GSK‐3 in mouse (B16BL6) and human (A2058) malignant melanoma cells, and thereby strongly inhibited their proliferation. DIF‐1 suppressed the canonical Wnt signaling pathway by lowering the expression levels of transcription factor 7‐like 2 and &bgr;‐catenin, key transcription factors in this pathway. DIF‐1 also inhibited cell migration and invasion, reducing the expression of matrix metalloproteinase‐2; however, this effect was not dependent on GSK‐3 activity. In a mouse lung tumor formation model, repeated oral administrations of DIF‐1 markedly reduced melanoma colony formation in the lung. These results suggest that DIF‐1 inhibits cell proliferation by a GSK‐3‐dependent mechanism and suppresses cell migration and invasion by a GSK‐3‐independent mechanism. Therefore, DIF‐1 may have a potential as a novel anti‐cancer agent for the treatment of malignant melanoma.

Inhibition of GSK-3β increases trabecular bone volume but not cortical bone volume in adenine-induced uremic mice with severe hyperparathyroidism

Patients with chronic kidney disease (CKD) are at increased risk for bone fractures compared with the general population. Repression of the Wnt/β‐catenin signaling pathway is associated with bone abnormalities. Inhibition of glycogen synthase kinase (GSK)‐3β, a critical component of the Wnt/β‐catenin signaling pathway, increases bone volume through accumulation of β‐catenin. It remains unknown whether inhibition of GSK‐3β increases bone volume in CKD. The present in vivo study examined the effects of GSK‐3β inhibition on bone volume in CKD mice. Wild‐type mice were divided into three groups. One group was fed a control diet (CNT) and the other two groups were fed a diet containing 0.2% adenine and given water with or without lithium chloride (LiCl), a GSK‐3 inhibitor (CKD, CKD+LiCl, respectively). GSK‐3β heterozygous knockout mice were fed a diet containing 0.2% adenine (CKD‐GSK‐3β+/−). After 6 weeks, trabecular and cortical bone volumes of the femur were analyzed using microcomputed tomography. CKD mice developed azotemia, hyperphosphatemia, and hyperparathyroidism, followed by a decrease in cortical bone volume without any change in trabecular bone volume. Serum levels of urea nitrogen, phosphate, and parathyroid hormone were comparable among the three groups of CKD mice. Trabecular bone volume increased in CKD‐GSK‐3β+/− and CKD+LiCl mice compared with CNT and CKD mice. However, there were no significant differences in cortical bone volume among the three groups of CKD mice. The results suggest that inhibition of GSK‐3β increases trabecular bone volume but not cortical bone volume in adenine‐induced uremic mice with uncontrolled hyperparathyroidism.

Inhibition of GSK-3 reduces prostaglandin E2 production by decreasing the expression levels of COX-2 and mPGES-1 in monocyte/macrophage lineage cells

Inflammatory stimuli induce prostaglandin E2 (PGE2) synthesis by upregulating cycloxgenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). Glycogen synthase kinase-3 (GSK-3) reportedly plays an important role in inflammatory reactions, whereas the role of this enzyme in inflammatory PGE2 production remains unclear. In the present study, therefore, we examined whether inhibition of GSK-3 can reduce inflammatory PGE2 production in vitro and in vivo. When macrophage-like cells differentiated from THP-1 were stimulated with lipopolysaccharide (LPS), PGE2 production and the expression levels of COX-2 and mPGES-1 were markedly elevated. GSK-3 inhibitors LiCl and SB216763 strongly suppressed their protein levels through inhibition of mRNA expressions. Subsequently, we examined the effect of GSK-3 inhibitors on nuclear factor κB (NF-κB) and early growth response-1 (Egr-1). The GSK-3 inhibitors had no significant effect on the NF-κB pathway, whereas they significantly decreased the expression level of Egr-1. Pharmacological and genetic inhibitions of GSK-3 also strongly suppressed PGE2 production in cultured peritoneal macrophages and in inflammatory air pouches made under the skin of living mice. These results suggested that GSK-3 plays a key role in PGE2 production by increasing COX-2 and mPGES-1 probably through Egr-1-mediated transcription and GSK-3 inhibitors may be potential as novel anti-inflammatory drugs.

Inorganic phosphate-induced impairment of osteoclast cell-cell fusion by the inhibition of AP-1-mediated DC-STAMP expression

Chronic kidney disease (CKD) causes hyperphosphatemia and secondary hyperparathyroidism, leading to several disorders of bone metabolism. Although high concentrations of extracellular inorganic phosphate (Pi) inhibit osteoclastogenesis, the molecular mechanism of this effect has not been fully understood. In the present study, therefore, we examined the effect of Pi on the differentiation of the osteoclast precursor RAW-D cells. Treatment with the receptor activator of nuclear factor-kappa B ligand induced the differentiation of RAW-D cells (osteoclastogenesis). However, Pi significantly weakened this effect, assessed by the tartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-positive multinucleated cells. Pi also reduced the expressions of nuclear factor of activated T-cell (NFAT) c1 and dendritic cell-specific transmembrane protein (DC-STAMP). Interestingly, the Pi-induced reduction of DC-STAMP gene promoter activity was lost when the activator protein 1 (AP-1) binding site was mutated. Since Pi strongly inhibited the expression of c-Fos which is the component of AP-1, the Pi-induced reduction of DC-STAMP expression was proposed to be mediated by the absence of c-Fos. These results suggested that hyperphosphatemia in the patients with CKD suppresses bone resorption by inhibiting osteoclastogenesis, and this impairs the regulation of bone metabolism.